Machine Learning-Supported Diagnosis of Small Blue Round Cell Sarcomas Using Targeted RNA Sequencing.

Small blue round cell sarcomas (SBRCSs) are a heterogeneous group of tumors with overlapping morphologic features but markedly varying prognosis. They are characterized by distinct chromosomal alterations, particularly rearrangements leading to gene fusions, whose detection currently represents the most reliable diagnostic marker. Ewing sarcomas are the most common SBRCSs, defined by gene fusions involving EWSR1 and transcription factors of the ETS family, and the most frequent non-EWSR1-rearranged SBRCSs harbor a CIC rearrangement. Unfortunately, currently the identification of CIC::DUX4 translocation events, the most common CIC rearrangement, is challenging. Here, we present a machine-learning approach to support SBRCS diagnosis that relies on gene expression profiles measured via targeted sequencing. The analyses on a curated cohort of 69 soft-tissue tumors showed markedly distinct expression patterns for SBRCS subgroups. A random forest classifier trained on Ewing sarcoma and CIC-rearranged cases predicted probabilities of being CIC-rearranged >0.9 for CIC-rearranged-like sarcomas and <0.6 for other SBRCSs. Testing on a retrospective cohort of 1335 routine diagnostic cases identified 15 candidate CIC-rearranged tumors with a probability >0.75, all of which were supported by expert histopathologic reassessment. Furthermore, the multigene random forest classifier appeared advantageous over using high ETV4 expression alone, previously proposed as a surrogate to identify CIC rearrangement. Taken together, the expression-based classifier can offer valuable support for SBRCS pathologic diagnosis.

Small cell osteosarcoma versus fusion-driven round cell sarcomas of bone: retrospective clinical, radiological, pathological, and (epi)genetic comparison with clinical implications.

Small cell osteosarcoma (SCOS), a variant of conventional high-grade osteosarcoma (COS), may mimic fusion-driven round cell sarcomas (FDRCS) by overlapping clinico-radiological and histomorphological/immunohistochemical characteristics, hampering accurate diagnosis and consequently proper therapy. We retrospectively analyzed decalcified formalin-fixed paraffin-embedded (FFPE) samples of 18 bone tumors primarily diagnosed as SCOS by methylation profiling, fusion gene analysis, and immunohistochemistry.In eight cases, the diagnosis of SCOS was maintained, and in 10 cases it was changed into FDRCS, including three Ewing sarcomas (EWSR1::FLI1 in two cases and no identified fusion gene in the third case), two sarcomas with BCOR alterations (KMT2D::BCOR, CCNB3::BCOR, respectively), three mesenchymal chondrosarcomas (HEY1::NCOA2 in two cases and one case with insufficient RNA quality), and two sclerosing epithelioid fibrosarcomas (FUS::CREBL3 and EWSR1 rearrangement, respectively).Histologically, SCOS usually possessed more pleomorphic cells in contrast to the FDRCS showing mainly monomorphic cellular features. However, osteoid was seen in the latter tumors as well, often associated with slight pleomorphism. Also, the immunohistochemical profile (CD99, SATB2, and BCOR) overlapped.Clinically and radiologically, similarities between SCOS and FDRCS were observed, with by imaging only minimal presence or lack of (mineralized) osteoid in most of the SCOSs.In conclusion, discrimination of SCOS, epigenetically related to COS, versus FDRCS of bone can be challenging but is important due to different biology and therefore therapeutic strategies. Methylation profiling is a reliable and robust diagnostic test especially on decalcified FFPE material. Subsequent fusion gene analysis and/or use of specific immunohistochemical surrogate markers can be used to substantiate the diagnosis.

Spontaneous expression of the CIC::DUX4 fusion oncoprotein from a conditional allele potently drives sarcoma formation in genetically engineered mice.

CIC::DUX4 sarcoma (CDS) is a rare but highly aggressive undifferentiated small round cell sarcoma driven by a fusion between the tumor suppressor Capicua (CIC) and DUX4. Currently, there are no effective treatments and efforts to identify and translate better therapies are limited by the scarcity of patient tumor samples and cell lines. To address this limitation, we generated three genetically engineered mouse models of CDS (Ch7CDS, Ai9CDS, and TOPCDS). Remarkably, chimeric mice from all three conditional models developed spontaneous soft tissue tumors and disseminated disease in the absence of Cre-recombinase. The penetrance of spontaneous (Cre-independent) tumor formation was complete irrespective of bi-allelic Cic function and the distance between adjacent loxP sites. Characterization of soft tissue and presumed metastatic tumors showed that they consistently expressed the CIC::DUX4 fusion protein and many downstream markers of the disease credentialing the models as CDS. In addition, tumor-derived cell lines were generated and ChIP-seq was preformed to map fusion-gene specific binding using an N-terminal HA epitope tag. These datasets, along with paired H3K27ac ChIP-sequencing maps, validate CIC::DUX4 as a neomorphic transcriptional activator. Moreover, they are consistent with a model where ETS family transcription factors are cooperative and redundant drivers of the core regulatory circuitry in CDS.

CIC-Rearranged Sarcoma.

CIC-rearranged sarcoma is a rare type of small round cell sarcoma. The tumors often affect the deep soft tissues of patients in a wide age range. They are highly aggressive, respond poorly to chemotherapy, and have a worse outcome than Ewing sarcoma. CIC-rearranged sarcoma has characteristic and recognizable histology, including lobulated growth, focal myxoid changes, round to epithelioid cells, and minimal variation of nuclear size and shape. Nuclear ETV4 and WT1 expression are useful immunohistochemical findings. CIC fusion can be demonstrated using various methods; however, even next-generation sequencing suffers from imperfect sensitivity, especially for CIC::DUX4.

Renal CIC-LEUTX rearranged sarcoma with multiple pulmonary metastases: a case report and literature review.

BACKGROUND: CIC-rearranged sarcomas (CRS) are a group of heterogeneous tumors which mostly occur in the soft tissues of limbs and trunk, and are highly invasive with poor prognosis. Here, we describe a rare case of CRS that occurred in the left kidney with a CIC-LEUTX rearrangement. CASE PRESENTATION: A 45-year-old male was admitted to hospital with a dry cough for more than two months without obvious cause. Physical examination and laboratory tests revealed no notable abnormality. The CT scan demonstrated a mass in the left kidney and multiple nodules in both lungs. The percutaneous core needle biopsy showed similar histomorphology and immunophenotype of small round cell malignant tumors. Genetic test revealed a CIC-LEUTX gene fusion. CONCLUSIONS: We present a rare primary renal CRS with multiple pulmonary metastases, and LEUTX is confirmed as the fusion partner of CIC gene for the first time in a renal case.

A Practical Approach to Small Round Cell Tumors Involving the Gastrointestinal Tract and Abdomen.

Small round cell neoplasms are diagnostically challenging owing to their clinical and pathologic overlap, necessitating use of large immunopanels and molecular analysis. Ewing sarcomas (ES) are the most common, but EWSR1 is translocated in several diverse neoplasms, some with round cell morphology. Molecular advances enable classification of many tumors previously termed 'atypical ES'. The current WHO Classification includes two new undifferentiated round cell sarcomas (with CIC or BCOR alterations), and a group of sarcomas in which EWSR1 partners with non-Ewing family transcription factor genes. This article reviews the spectrum of small round cell sarcomas within the gastrointestinal tract and abdomen.

[Clinical and morphological characteristics of Ewing's sarcoma and the algorithm for diagnosing undifferentiated round cell sarcomas]. / Kliniko-morfologicheskaya kharakteristika sarkomy Yuinga i algoritm diagnostiki nedifferentsirovannykh kruglokletochnykh sarkom.

BACKGROUND: The group of undifferentiated round cell sarcomas, according to the World Health Organization Classification, in addition to Ewing's sarcoma (ES), includes round cell sarcoma with rearrangement of the EWSR1 gene with partners not from the ETS gene family, sarcoma with BCOR gene alterations, CIC -rearranged sarcoma. Despite the fact that all tumors have clear histological and immunological criteria, their diagnosis can be difficult, given the fact that there are overlapping variants of the morphological picture and immunophenotype both within the group and with other round cell tumors. OBJECTIVE: Present a comparative analysis of genetically verified ES, sarcoma with BCOR gene alterations and CIC-rearranged sarcoma. MATERIAL AND METHODS: A comparative study of biopsy specimens of bones, soft tissues and internal organs was carried out in 118 patients with ES, 10 with BCOR gene alterations and 8 with CIC-rearranged sarcomas. All cases were genetically verified. The following research methods were used: histological, immunohistochemical, RT-PCR, RNA sequencing and FISH. RESULTS: Within our cohort, it was shown that ES predominantly affects bones, while soft tissue localization is more typical for the other two undifferentiated round cell sarcomas. Histologically, in the overwhelming majority of cases, ES is characterized by a monomorphic round-cell structure; on the contrary, heterogeneous structure is typical for sarcoma with alterations of the BCOR gene, CIC-rearranged sarcoma. High sensitivity and specificity of CD99/NKX2.2 co-expression for ES, BCOR/SATB2/TLE1 for sarcoma with BCOR gene alterations, high specificity and low sensitivity of WT1/ETV4 co-expression for CIC-rearranged sarcoma was shown. CONCLUSION: For the differential diagnosis of undifferentiated round-cell sarcomas, it is necessary to take into account the clinical, morphology when compared with the data of the IHC study, and verification by molecular genetic methods is necessary to improve the accuracy of diagnosis.

[CIC-rearranged sarcoma: a case report and literature review]. / CIC-perestroennaya sarkoma: klinicheskoe nablyudenie i obzor literatury.

CIC-rearranged sarcoma is a rare and extremely aggressive tumor that occurs mainly in soft tissues. Despite the fact that identification of a characteristic genetic rearrangement is necessary to verify the diagnosis, in most cases, the correct diagnosis can be made by comparing histological signs and a characteristic immunophenotype, which greatly speeds up the diagnosis. The article describes a case of CIC-rearranged sarcoma in a 14-year girl with the successful application of the CWS-2009 treatment protocol.

Clinicopathological features of five cases of CIC::DUX4 positive sarcomas, including literature review.

According to the recent World Health Organization (WHO) classification, CIC-rearranged sarcomas, including CIC::DUX4-positive sarcomas constitute an aggressive subtype of undifferentiated round cell sarcomas. There is a single study on these tumors from our subcontinent. We present clinicopathological features of 5 additional cases of this tumor entity, including literature review. Thirty-nine undifferentiated round cell sarcomas, excluding Ewing sarcomas (ES), were tested for CIC::DUX4 fusion, including Type I (165 base pair size) and II (230 bp) by reverse transcription-polymerase chain reaction. Twenty-five of those tumors were tested for EWSR1 gene rearrangement, 5 for SS18 and 4 for SS18::SSX fusion, and were negative for those tests. Five tumors (12.8 %) were positive for CIC::DUX4(Type II) fusion. Five CIC:: DUX4-positive sarcomas occurred in 4 males and one female; of 25-43 years of age, in soft tissues, including thigh (n = 2), chest wall (n = 1), iliac region (n = 1) and foot (n = 1). Tumor size varied from 2.2 to 19 cm. Microscopically, the tumors were predominantly composed of nodules and sheets of malignant round to epithelioid cells, including "rhabdoid-like" (n = 2) and spindle-shaped (n = 2) with eosinophilic to vacuolated cytoplasm (4/5), distinct nucleoli (4/5), brisk mitoses, focal myxoid to hyalinised stroma (4/5) and necrosis (5/5). Immunohistochemically, tumor cells were positive for WT1 (5/5), calretinin (3/4), pan-keratin (1/4), CD99/MIC2 ("dot-like" to cytoplasmic membranous) (4/4), while negative for desmin (0/4), S100P (0/4), and NKX2.2 (0/5). INI1/SMARCB1 was retained (3/3). All patients underwent excision with adjuvant radiotherapy and chemotherapy (Ewing sarcoma regimen). A single patient developed recurrence, and 2 developed pulmonary metastasis, including one with brain metastasis. CIC:: DUX4-positive sarcomas are ultra-rare tumors, that mainly occur in the soft tissues and in young adult patients. Histopathologically, these tumors display a wide spectrum, including round to epithelioid cells, variable amount of cytoplasmic vacuolization and myxoid stroma with necrosis. Immunohistochemically, these tumors express WT1 and calretinin. Despite adjuvant therapies, these tumors have dismal outcomes, especially in large-sized tumors. CIC::DUX4-positive sarcomas need to be differentiated from their histopathological mimics, including ES, in view of significant treatment-related implications.

CIC-rearranged sarcoma presenting with superior vena cava syndrome: case report.

CIC-rearranged sarcomas are rare mesenchymal neoplasms belonging to the family of undifferentiated small round cell sarcomas. This report details the case of a 45-year-old man presenting with symptoms of mediastinal compression, radiological diagnosis of a mediastinal mass and rapid evolution to full-blown superior vena cava syndrome. The emergency was successfully managed with a pharmacological approach. Formulation of a pathological diagnosis of CIC-rearranged sarcoma was initially supported by fluorescence in situ hybridisation findings and later validated by next-generation sequencing, which showed CIC-DUX4 gene fusion. A chemotherapy regimen was started with immediate benefits for the patient. The spectrum of pathological entities able to cause superior vena cava syndrome is wide, and recognition of rare causes is important to tailor the therapeutic approach to the specific disease. This is, to the best of our knowledge, the first report of CIC-rearranged sarcoma presenting with superior vena cava syndrome.

A global collaboRAtive study of CIC-rearranged, BCOR::CCNB3-rearranged and other ultra-rare unclassified undifferentiated small round cell sarcomas (GRACefUl).

BACKGROUND: Undifferentiated small round cell sarcomas (URCSs) represent a diagnostic challenge, and their optimal treatment is unknown. We aimed to define the clinical characteristics, treatment, and outcome of URCS patients. METHODS: URCS patients treated from 1983 to 2019 at 21 worldwide sarcoma reference centres were retrospectively identified. Based on molecular assessment, cases were classified as follows: (1) CIC-rearranged round cell sarcomas, (2) BCOR::CCNB3-rearranged round cell sarcomas, (3) unclassified URCSs. Treatment, prognostic factors and outcome were reviewed. RESULTS: In total, 148 patients were identified [88/148 (60%) CIC-rearranged sarcoma (median age 32 years, range 7-78), 33/148 (22%) BCOR::CCNB3-rearranged (median age 17 years, range 5-91), and 27/148 (18%) unclassified URCSs (median age 37 years, range 4-70)]. One hundred-one (68.2%) cases presented with localised disease; 47 (31.8%) had metastases at diagnosis. Male prevalence, younger age, bone primary site, and a low rate of synchronous metastases were observed in BCOR::CCNB3-rearranged cases. Local treatment was surgery in 67/148 (45%) patients, and surgery + radiotherapy in 52/148 (35%). Chemotherapy was given to 122/148 (82%) patients. At a 42.7-month median follow-up, the 3-year overall survival (OS) was 92.2% (95% CI 71.5-98.0) in BCOR::CCNB3 patients, 39.6% (95% CI 27.7-51.3) in CIC-rearranged sarcomas, and 78.7% in unclassified URCSs (95% CI 56.1-90.6; p < 0.0001). CONCLUSIONS: This study is the largest conducted in URCS and confirms major differences in outcomes between URCS subtypes. A full molecular assessment should be undertaken when a diagnosis of URCS is suspected. Prospective studies are needed to better define the optimal treatment strategy in each URCS subtype.

Expanding the Molecular Diversity of CIC-Rearranged Sarcomas With Novel and Very Rare Partners.

Capicua transcriptional repressor (CIC)-rearranged sarcoma represents a distinct pathologic entity and constitutes the second most prevalent category of undifferentiated round cell sarcomas (URCSs) after Ewing sarcoma. The 2 most common translocations are t(4;19) and t(10;19), resulting in CIC fusions with either DUX4 and DUX4L paralog, respectively; however, other rare variant fusions have also been reported. In this study, we expand the molecular spectrum of CIC-gene partners, reporting on 5 cases of URCSs showing CIC fusions with AXL, CITED1, SYK, and LEUTX by targeted RNA or DNA sequencing. There were 4 female patients and 1 male patient with a wide age range (12-70 years; median, 36 years). Four cases occurred in the deep soft tissues (lower extremity, 3; neck, 1) and 1 case in the central nervous system (midbrain/thalamus). All cases showed similar histologic findings within the spectrum of URCSs. Immunohistochemistry, showed variable positivity for ETV4 in 4 of the 4 cases and positive results for ERG in 3 of the 4 cases and for WT1 in 1 of the 4 cases. CD31 showed positivity in 2 of the 3 cases, including one coexpressing ERG. Unsupervised clustering of methylation profiles by T-distributed stochastic neighborhood embedding performed in 4 cases showed that all clustered tightly together and along the CIC sarcoma methylation class. RNA-sequencing data showed consistent upregulation of ETV1 and ETV4 mRNA in all cases examined, at similar levels to CIC::DUX4 URCSs. Our study expands the molecular diversity of CIC-rearranged URCSs to include novel and rare partners, providing morphologic, immunohistochemical, gene expression, and methylation evidence supporting their classification within the family of tumors harboring the more common DUX4/DUX4L partner genes.

Approach to Primary Vertebral Tumors in the Light of the 2020 Updated World Health Organization Classification of Bone Tumors.

AIM: To define a new approach for primary vertebral tumors by discussing them in the light of 2020 World Health Organization (WHO) classification of bone tumors. MATERIAL AND METHODS: In this study, we have discussed primary vertebral tumors in light of the 2020 Updated WHO Classification of Soft Tissue and Bone Tumors. RESULTS: Chondroblastoma and chondromyxoid fibroma has been classified in the benign category, while synovial chondromatosis has been moved from the benign category to the intermediate category. In the updated classification, grade I chondrosarcoma has been classified in the malignant category with grade II?III chondrosarcomas. Minor amendments have been made for osteosarcoma subtypes. Neoplasms of undetermined origin, such as aneurysmal cysts, simple bone cysts, fibrosis dysplasia, and osteofibrous dysplasia, have been classified as neoplastic lesions. Chordomas have been classified into ?not otherwise stated,? poorly differentiated chordomas, and dedifferentiated chordomas. Ewing?s sarcomas have been classified in a separate section for undifferentiated, small, round cell sarcomas of the bone and soft tissue. In this section, three distinct subsets different from Ewing?s sarcoma have been discussed. CIC-rearranged sarcoma, BCOR-rearranged sarcoma, and round cell sarcomas with EWSR1 gene fusion with non-ETS family members. CONCLUSION: In this study, we have reviewed the new classifications and discussed their effect on decision making in spinal oncologic surgery.

Ewing and Ewing-like sarcomas: A morphological guide through genetically-defined entities.

The fifth edition of the World Health Organization classification of soft tissue and bone tumors redefined Ewing sarcoma by fusions between EWSR1/FUS and ETS family of transcription factors, and recognized three tumor groups among Ewing-like sarcoma: CIC-rearranged sarcoma, sarcoma with BCOR genetic alterations, and round cell sarcoma with EWSR1::non-ETS fusions. Although this classification underscores the critical role of molecular genetics in the diagnosis of small round cell sarcoma, each entry is recognized as a specific entity not only because they have different genetics but because their phenotypes are distinct and reasonably robust to support the diagnosis. This review focuses on the morphological aspects of Ewing sarcoma and a subset of Ewing-like sarcomas (CIC-rearranged sarcoma, BCOR-associated sarcoma, and EWSR1::NFATC2 sarcoma) for which phenotypic characteristics have been well established. Classic histological findings, uncommon variations, and recurrent diagnostic pitfalls are addressed, along with the utility of recently developed immunohistochemical markers (NKX2.2, PAX7, ETV4, BCOR, CCNB3, and NKX3.1). Phenotypic expertise would significantly expedite the diagnostic process and complement (or sometimes outperform) genetic testing, even in well-resourced settings. Morphological knowledge plays an even more substantial role in facilities that do not have easy access to molecular testing.

[CIC-rearranged sarcoma with rhabdoid features: a clinicopathological analysis].

Objective: To investigate the histopathologic, immunohistochemical, molecular genetic characteristics of CIC-rearranged sarcoma (CRS) with rhabdoid features. Methods: The clinical and pathologic data of two cases of CRS diagnosed between 2019 and 2021 at the Department of Pathology, Jiangsu Province Hospital were analyzed. Immunohistochemical study and fluorescence in situ hybridization (FISH) were performed. The relevant literature was reviewed. Results: Both patients were female, one was 58 years old, with tumor located in left thigh; the other was 43 years old, with tumor located in left pelvic cavity. Microscopically, both tumors were composed of small to medium-sized round, oval cells, arranged in nodules or sheets. The tumor cells showed irregular nuclear outline, coarse chromatin with prominent nucleoli and brisk mitotic activity. Both cases showed rhabdoid phenotype with myxoid stromal changes. Immunohistochemically, both cases were positive for CD99 and c-myc. High WT1 reactivity was seen in classic area, with low reactivity in rhabdoid area. There was no INI1 lost in both cases. Both were negative for NKX2.2 and NKX3.1. By FISH both cases demonstrated convincing break-apart signals of CIC gene. One patient died of disease after 1 month, and the other died of disease after 3 months. Conclusions: CRS is a small round cell undifferentiated sarcoma of the bone and soft tissue defined by molecular genetic characteristics, and may show atypical morphologic and immunophenotypic characteristics such as rhabdoid features. A correct understanding of its rare morphologic and immunophenotypic characteristics, combined with molecular pathologic detection, is conducive to correct diagnosis.

Small round cell sarcomas.

Undifferentiated small round cell sarcomas (SRCSs) of bone and soft tissue comprise a heterogeneous group of highly aggressive tumours associated with a poor prognosis, especially in metastatic disease. SRCS entities mainly occur in the third decade of life and can exhibit striking disparities regarding preferentially affected sex and tumour localization. SRCSs comprise new entities defined by specific genetic abnormalities, namely EWSR1-non-ETS fusions, CIC-rearrangements or BCOR genetic alterations, as well as EWSR1-ETS fusions in the prototypic SRCS Ewing sarcoma. These gene fusions mainly encode aberrant oncogenic transcription factors that massively rewire the transcriptome and epigenome of the as yet unknown cell or cells of origin. Additional mutations or copy number variants are rare at diagnosis and, depending on the tumour entity, may involve TP53, CDKN2A and others. Histologically, these lesions consist of small round cells expressing variable levels of CD99 and specific marker proteins, including cyclin B3, ETV4, WT1, NKX3-1 and aggrecan, depending on the entity. Besides locoregional treatment that should follow standard protocols for sarcoma management, (neo)adjuvant treatment is as yet ill-defined but generally follows that of Ewing sarcoma and is associated with adverse effects that might compromise quality of life. Emerging studies on the molecular mechanisms of SRCSs and the development of genetically engineered animal models hold promise for improvements in early detection, disease monitoring, treatment-related toxicity, overall survival and quality of life.

A case of CIC-rearranged sarcoma with CIC-LEUTX gene fusion in spinal cord.

A 16-year-old male was admitted to the hospital for weakness of both lower extremities. Magnetic resonance imaging revealed an intraspinal extramedullary subdural mass at the thoracic 9 level. Microscopically, the tumor cells were small to medium sized and round to ovoid in shape. They were distributed in diffuse sheets or showed nodular appearance. The nucleus of the tumor had mild-to-moderate atypia, with vesicular chromatin and prominent nucleoli. A smaller proportion of tumor cells demonstrated rhabdoid morphology. Focal myxoid stromal change was present, in which tumor cells exhibited spindle shapes. Approximately two mitoses were counted per 10 high-power fields. No necrosis was observed. The tumor cells were focal positive for CD99; multifocal positive for WT1; diffuse positive for nestin, synaptophysin, and D2-40; partial positive for GFAP; focal positive for desmin and SSTR2; and scattered positive for S-100 protein. The Ki-67 labeling index was approximately 20%. Genetic testing revealed CIC-LEUTX gene fusion. Considering the patient's history, clinical data, pathological findings and genetic findings, we rendered a rare tumor named CIC-rearranged sarcoma with CIC-LEUTX gene fusion.

Novel ATXN1/ATXN1L::NUTM2A fusions identified in aggressive infant sarcomas with gene expression and methylation patterns similar to CIC-rearranged sarcoma.

CIC-rearranged sarcomas are newly defined undifferentiated soft tissue tumors with CIC-associated fusions, and dismal prognosis. CIC fusions activate PEA3 family genes, ETV1/4/5, leading to tumorigenesis and progression. We report two high-grade CNS sarcomas of unclear histological diagnosis and one disseminated tumor of unknown origin with novel fusions and similar gene-expression/methylation patterns without CIC rearrangement. All three patients were infants with aggressive diseases, and two experienced rapid disease deterioration and death. Whole-transcriptome sequencing identified an ATXN1-NUTM2A fusion in the two CNS tumors and an ATXN1L-NUTM2A fusion in case 3. ETV1/4/5 and WT1 overexpression were observed in all three cases. Methylation analyses predicted CIC-rearranged sarcoma for all cases. Retrospective IHC staining on case 2 demonstrated ETV4 and WT1 overexpression. ATXN1 and ATXN1L interact with CIC forming a transcription repressor complex. We propose that ATXN1/ATXN1L-associated fusions disrupt their interaction with CIC and decrease the transcription repressor complex, leading to downstream PEA3 family gene overexpression. These three cases with novel ATXN1/ATXN1L-associated fusions and features of CIC-rearranged sarcomas may further expand the scope of "CIC-rearranged" sarcomas to include non-CIC rearrangements. Additional cases are needed to demonstrate if ATXN1/ATXN1L-NUTM2A fusions are associated with younger age and more aggressive diseases.

Central nervous system sarcoma with ATXN1::DUX4 fusion expands the concept of CIC-rearranged sarcoma.

CIC-rearranged sarcoma is a high-grade sarcoma, most often harboring CIC::DUX4 fusion, and is characterized by a distinct round cell histology, co-expression of ETV4 and WT1, and a specific DNA methylation class. Herein, we report a brain tumor with ATXN1::DUX4 that had an indistinguishable phenotype and DNA methylation profile from CIC-rearranged sarcoma. A 40-year-old man presented with a 5 cm hemorrhagic mass in the right frontal lobe of the cerebrum. The tumor was resected and histologically showed a dense proliferation of relatively monomorphic round cells with multifocal myxoid changes. Immunohistochemically, the tumor was diffusely positive for ETV4, WT1, and DUX4. Through classic histomorphology and immunoprofile, the tumor was provisionally diagnosed as CIC-rearranged sarcoma. However, no CIC fusions or mutations were identified using CIC break-apart fluorescence in situ hybridization (FISH) or FoundationOne CDx. Despite multiple surgeries and adjuvant chemoradiation therapy, the patient succumbed 16 months after presentation. RNA exome sequencing detected an in-frame intraexonic ATXN1 (exon 9)::DUX4 (exon 1) fusion, which was validated by reverse transcription-polymerase chain reaction and ATXN1 FISH assay. Upon DNA methylation analysis, the tumor matched with CIC-rearranged sarcoma both by the Deutsche Krebsforschungszentrum classifier and t-distributed stochastic neighbor embedding. Along with a recent report of a similar pediatric brain tumor, the present case suggests that ATXN1::DUX4 is a recurrent alternative molecular event in the sarcoma type that is presently defined by CIC rearrangement, which prompts an expansion of the tumor concept.